GSM6730582: WCE, C57, Liver, Fructose; Mus musculus; ChIP-Seq
25-30 mg of frozen liver tissue were quickly minced with razor blades in PBS at room temperature. Tissue was crosslinked with 0.5 M disuccinimidyl glutarate in PBS for 45 min at room temperature, followed by second fixation with 1% formaldehyde in Fixing Buffer A (Covaris) for 5 min at room temperature. Crosslinking was stopped by Quenching Buffer E (Covaris). After washing, nuclei were isolated by Dounce homogenization followed by centrifugation. After washing and centrifugation, the nuclear pellet was resuspended in cold 0.25% SDS Shearing Buffer (Covaris). Chromatin was sheared in 1 ml AFA milliTUBEs (Covaris) using Covaris S220X focused ultrasonicator with the following parameters: peak incident power 140W, duty factor 5%, cycles per burst 200 for 12 min. The sheared chromatin was centrifuged at 13,000rpm for 10min at 4°C to remove debris, and a 10 ul aliquot was de-crosslinked and used for quantification with Qubit (Thermo Fisher Scientific). Sheared chromatin (1.5 – 3 μg) was diluted in ChIP dilution buffer (16.7 mM Tris [pH8], 1.2 mM EDTA, 25 mM NaCl, 1.1% Triton X-100, 0.01% SDS), and 1 μg of  ChREBP antibody (Novus, NB400-135) or control rabbit IgG was added, followed by overnight incubation at 4°C. The reactions were then incubated for 1h at 4°C with protein A/G dynabeads (Invitrogen) pre-blocked in PBS/0.5% BSA/0.5% Tween. Beads were then washed in low salt wash buffer (20 mM Tris [pH8], 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), high salt wash buffer (20 mM Tris [pH8], 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), LiCl wash buffer (10 mM Tris [pH8], 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 250 mM LiCl) and TE buffer (10 mM Tris [pH8], 1 mM EDTA) and eluted and reverse cross-linked in elution buffer (10 mM Tris [pH8], 5 mM EDTA, 0.1% SDS, 300 mM NaCl, 0.8 mg/ml proteinase K, 10 μg/ml RNase A) by incubating at 65°C for 10 hours. Next, DNA was extracted using AMPure XP beads following the manufacturer's manual and quantified by Qubit (Thermo Fisher Scientific). Immunoprecipitated chromatin was pooled by genotype and gavage condition for further analysis. Sequencing libraries were constructed using the “on-bead” preparation method as previously described (Kang, S., Tsai, L.T., Zhou, Y., Evertts, A., Xu, S., Griffin, M.J., Issner, R., Whitton, H.J., Garcia, B.A., Epstein, C.B., et al. (2015)). Briefly, either 5ng (ChREBP) or 10ng (WCE input) of immunopreciptated chromatin were used as input for library construction. We performed end repair/ phosphorylation using the End-It DNA End-Repair Kit (Epicentre), A-tailing using the Klenow Fragment (NEB M0212) and index adaptor ligation using Quick Ligase (NEB M2200). AMPure XP beads were used for isolation of DNA per manufacturer protocol. After ligation, DNA was eluted and amplified by PCR for 12 (WCE) or 16 (ChREBP) cycles using the PfuUltra II Hotstart PCR Master Mix (Agilent 600850). DNA sized between 250 and 600bp was selected by gel electrophoresis and extraction using E-Gel EX Agarose Gels (Invitrogen) and MinElute Gel Extraction kits (Qiagen). Libraries were analyzed using the Qubit and Agilent Bioanalyzer, pooled at a final concentration of 12pM for sequencing.